Raman spectroscopy and Raman microscopy are conventionally used to measure the vibration levels of molecules in solution, in the gas phase, solid phase or on surfaces. In a Raman scattering process, a molecule can absorb energy from an incident photon, with scattered photons being down-shifted in energy (Stokes shift). Scattered photons can also gain energy from populated vibronic states of the molecule and are up-shifted in energy (Anti-Stokes shift). The ensemble of Stokes-shifted lines are referred to as vibronic molecular fingerprint of the molecule.
Disadvantageously, in conventional Raman spectroscopy, fluorescent emission can coincide with the Stokes-shifted Raman spectrum to be measured. Short laser pulses and time-gated signal detection may be employed to alleviate the adverse effects caused by fluorescence. Spectra of complex organic molecules in mixtures may also overlap, making it difficult to determine an association between the measured spectra and the molecules of interest. The low efficiency of scattering <10−6 for non-resonant scattering, and background scattering may also require concentrations of molecules of interest in excess of 1% of scattering molecules which may be impractical.
Coherent Anti-Stokes Raman Spectroscopy (CARS) has recently been used to address many of the aforementioned problems. As illustrated in FIG. 1, CARS is a four-wave mixing process using three photons (ωp1, ωs, ωp2) to prepare a vibronic or rotational state, stimulating the resulting in the emission of a fourth photon (ω3) that is blue-shifted relative to the other three photons. The first photon (ωp1) is called the pump photon, while the second photon (ωs) is called the Stokes photon. The third photon (ωp2), the probe photon, drives the electron to a virtual level from which the fourth photon (ω3) is emitted as the electron returns to the ground state. Since vibronic excited states relax on a picosecond to sub-picosecond time scale, the process is most efficient when activated by sub-picosecond laser pulses. The signal strength is greatly enhanced over conventional Raman spectroscopy because the CARS process is a stimulated process and increases as the square of the laser intensity.
It has recently been reported by Oron et al. (Physical Review Letters Vol. 90, No. 21, pp. 213902-1 to -4 (2003)) that all three stimulating CARS photons (ωp1, ωs, ωp2) can be supplied by a single laser pulse and that an incoherent CARS background can be substantially reduced by controlling the laser polarization and the phase of arrival of the photons from the pulse at the molecule.
FIG. 2 shows a conventional single laser setup 20 for CARS spectroscopy which includes a phase and polarization controller 24. A laser 22, for example, a Ti: Sapphire laser, generates a femtosecond laser pulse. The laser pulse then passes through a spectral phase/pulse shaper 24 which can include, for example, a liquid crystal spectral light modulator (SLM) 244 and two gratings 242 and 246 that spectrally disperse and recombine/compress the pulse for laser 22. Pulse shaper 24 can also control the phase and polarization of the laser radiation emitted by laser 22. The shaped pulse exiting the spectral phase/pulse shaper 24 is then focused onto the sample 25, and the CARS signal is measured, for example, with a dispersive spectrometer 26 or a monochromator 26 with a photodetector, such as a photomultiplier tube, and analyzed in spectral analyzer 27. The phase/polarization controller 24 can be controlled by, for example, the spectral analyzer 27 via a data and control line 28. Although detection of the CARS signal is shown in transmission, a back scattered signal can also be detected.
However, the conventional CARS spectroscopy may not be able to associate, for example, in complex mixtures of molecules, the various Raman lines with the fingerprint of specific constituent molecules. This molecular spectral dissection is presently accomplished with chemometric spectral analysis algorithms that applies mathematical and statistical techniques to the analysis of complex data. However, this approach tends to be limited to less than ten overlapping fingerprints and may require concentrations of the constituent molecules of greater than 1% in solutes.
It would therefore be desirable to overcome the shortcomings of the conventional Raman measurement techniques and analytical methods, which would allow a spectroscopic analysis of complex mixtures of organic molecules and an unambiguous determination of the constituents.